The Phytophthora, which literally means plant destroyer, is a genus of pathogens whose name was coined in 1861 by Anton de Barry when he showed that a species of Phytophthora was responsible for the Irish potato famine (Large, The advance of the fungi. Jonathan Cape, Ltd., London (1940)). The Phytophthora genus includes more than 65 plant pathogenic oomycetes species. These pathogens cause devastating diseases in numerous crops, ornamentals and native plants. The Phytophthora pathogens have an enormous impact on agriculture. (See, e.g., Govers and Gijzen, Molecular Plant-Microbe Interactions, 19:1295-1301 (2006)).
A recently emerged Phytophthora species, Phytophthora ramorum, is the causal agent for oak sudden death and is responsible for extensive mortality of coast live oak (Quercus agrifolia) and tan oak (Lithocarpus densiflorus) in northwest California (Rizzo et al., Plant Disease, 86:205-214. (2002); Rizzo et al., Ann. Rev. of Phytopathology, 43:309-335 (2005); Grünwald et al. Ann. Rev. of Phytopathology, 43:171-190 (2008)). P. ramorum additionally causes foliar lesions and twig dieback (Ramorum blight) on hosts in over 40 plant genera including many common ornamentals (Rizzo et al., Ann. Rev. of Phytopathology, 43:309-335 (2005). Further, the infestation of nursery stock has provided a mechanism for long-distance dispersal and quarantine efforts have led to large economic losses by the nursery industry in North American and Europe. (See, e.g., Goss et al., Molecular Ecology, 18:1161-1174 (2009).)
There are three clonal lineages of P. ramorum, within which genetic variation has only been observed at rapidly evolving microsatellites (Ivors et al., Molecular Ecology, 15:1493-1505 (2006)). Support for grouping isolates into three lineages comes from AFLP, microsatellites, and mitochondrial sequence data and is consistent across markers (Ivors et al., Molecular Ecology, 108:378-392 (2004); Ivors et al., Molecular Ecology, 15:1493-1505 (2006); Prospero et al., Molecular Ecology, 16:2958-2973 (2007); Martin, Current Genetics, 54:23-34(2008)). The EU1 lineage is responsible for all infestations in Europe but has also been found in nurseries on the West Coast of the USA. The NA1 genotype is the cause of the wildland epidemics in California and the southwest corner of Oregon and is also seen in nursery populations (Prospero et al., Molecular Ecology, 16:2958-2973 (2007)). The third genotype is the NA2 genotype and has only been observed in a limited number of nurseries. (See, e.g., Goss, et al., Molecular Ecology, 18:1161-1174 (2009).)
P. ramorum is self-sterile and sexual reproduction must occur between individual organisms of different mating types. The EU1 genotype is largely the A1 mating type and all tested NA1 and NA2 isolates have been A2 (Ivors et al., Molecular Ecology, 15:1493-1505 (2006)). The two mating types have been brought together by the nursery trade (Grünwald et al., Plant Disease, 92:314-314 (2008)), facilitating pathogen spread, and have both been detected in a California creek (Frankel, Australasian Plant Pathology, 37:19-25 (2008)). The closest known relatives of P. ramorum are P. lateralis, P. foliorum, and P. hibernalis, which together make up Phytophthora clade 8c (Blair et al., Fungal Genetics and Biology, 45:266-277 (2008)). (See, e.g., Goss, et al., Molecular Ecology, 18:1161-1174 (2009).)
Due to the devastating effects P. ramorum exhibits with respect to numerous plant species, selective detection of P. ramorum would prove highly useful and economically beneficial. The methods of the present invention provide for detection of indole-3-glycerol-phosphate synthase N-5′-phosphoribosyl anthranilate isomerase (trp1) in Phytophthora ramorum using isothermal amplification methods. The detection methods described allow for specific detection of the P. ramorum species of the Phytophthora genus even in the presence of closely related species.
In 1991, the TRP1 gene was isolated from Phytophthora, specifically Phytophthora parasitica, and shown to encode indole-3-glycerol-phosphate synthase N-5′-phosphoribosyl anthranilate isomerase (trp1, also known as IGPS-PRAI) (Karlovsky and Prell, Gene, 109:161-165 (1991)). The TRP1 gene encodes the bifunctional indole-3-glycerol-phosphate synthase N-5′-phosphoribosyl anthranilate isomerase enzyme (Karlovsky and Prell, Gene, 109:161-165 (1991). The bifunctional trp1 enzyme contains both indole-3-glycerol-phosphate synthase (IGPS) and phosphoribosyl anthranilate isomerase (PRAI) activities and is involved in tryptophan biosynthesis (Karlovsky and Prell, Gene, 109:161-165 (1991); Stryer, Biochemistry, 4th Edition, New York, 1975, 1981, 1988, 1995).
The trp1 gene is found in many Phytophthora species, as well as other bacterial and fungal species (Karlovsky and Prell, Gene, 109:161-165 (1991)). However, upon experimental examination of samples from many closely related Phytophthora species, the nucleic acid amplification methods of the present invention allow for detection of P. ramorum while not detecting any other Phytophthora species.
Any methods known to one of skill in art for amplification of polynucleotides can be employed with the methods of the present invention. However, traditional PCR methods require high temperatures to separate DNA strands and lower temperatures to allow primer binding, and as such require the reaction mixture and reaction chamber to cycle through hot and cold phases. Thus, traditional PCR methodologies require sophisticated and expensive equipment for precisely controlling the temperature during the amplification reaction period.
Alternatively, isothermal amplification methods exhibit many benefits over traditional PCR amplification methods. One such isothermal method is recombinase polymerase amplification (RPA), developed by Piepenburg, Armes and colleagues (Piepenburg et al., PLOS Biology, 4(7):1115-1121 (2006)). RPA employs recombinase-primer complexes that scan double-stranded DNA and facilitate exchange at cognate sites, allowing binding of opposing oligonucleotides primers to template DNA and subsequent extension by DNA polymerase (Piepenburg et al., PLOS Biology, 4(7):1115-1121 (2006)). This system allows for amplification of DNA without the need for precise temperature regulation or expensive and sophisticated equipment.